1 Dilute and filter sample 2 Prepare spin cartridge 3 Apply sample 4 Wash and collect flow-through F1

5 Wash and collectflow-through F2

6 Prepare for elution 7 Elute bound fraction 8 Re-equilibration 9 Analyze F1 + F2

F1 F1 F2

F1 + F2

Fill 5-mL Luer-Lock plastic syringe with2-mL Buffer B and attach to Luer-Lockadapter. Slowly push Buffer B throughcartridge to elute bound proteins intonew collection tube. Save eluant withtargeted high-abundant proteins foranalysis or discard.

Remove cartridge cap and plug, attachLuer-Lock adapter to cartridge, draw 4 mL of Buffer A into syringe and dis-pense through cartridge via Luer-Lock,remove excess Buffer A from top ofresin bed with transfer pipette.

Dilute 7–10-µL human serum sample to200 µL with Buffer A. Consult cartridgecertificate for true sample capacity.Filter through 0.22-µm spin filter.

Remove Luer-Lock adapter and add200-µL diluted serum sample. Cap car-tridge loosely or leave open. Place in1.5-mL collection tube labeled “Flow-through fraction 1” (F1). Centrifuge 1.5 min at 100 × g.

Add 400-µL Buffer A. Centrifuge 2.5 min at 100 × g. Collect in F1 tube.

Place spin cartridge in new collectiontube labeled “Flow-through fraction 2”(F2). Add 400-µL Buffer A. Centrifuge2.5 min at 100 × g. Collect in F2 tube.

Remove spin cartridge from F2 tubeand attach Luer-Lock adapter tightly totop of cartridge.

Fill new 5-mL plastic syringe with 4-mL Buffer A and attach to Luer-Lockadapter. Slowly push Buffer A throughcartridge to re-equilibrate the cartridgefor the next sample or store wettedwith Buffer A (at 4 °C). Recap bothends for storage.

Fractions F1 and F2 can be analyzedindividually or combined. Concentrateand analyze these fractions containinglow-abundant proteins.

For more detailed instructions or informationon accessories, refer to theAgilent Multiple Affinity

Removal Spin CartridgeInstruction Guide

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Visit www.agilent.com/chem/bioreagents formore products that will help make your proteomics research more efficient:

- Multiple Affinity Removal LC columns for automated serum/plasma processing

- Lys Tag 4H reagent for improving MALDI-MSanalysis of peptides and proteins

- Tryptic digestion kit for digesting gel-isolated proteins

- C18 Cleanup Pipette Tips and Spin Tubes for purifying proteomics samples

- Spin filters and concentrators for cleaning, concentrating, and buffer-exchanging samples

- MALDI-MS matrices and peptide/protein standards for your proteomics studies

Protein In-Gel Tryptic Digestion Kit

Agilent shall not be liable for errors contained herein orfor incidental or consequential damages in connectionwith the furnishing, performance, or use of this material.

Information, descriptions, and specifications in thispublication are subject to change without notice.

© Agilent Technologies, Inc. 2004

Printed in the USAOctober 8, 20045188-5255EN

Multiple AffinityRemoval LC column

Agilent Multiple Affinity Removal Spin CartridgePart Number 5188-5230

Quick Reference Guide

For Human Serum

Lys Tag 4HReagent Kit

Cleanup C18 Pipette Tips

For More Information

For more information on our products and services, visit our Web site at www.agilent.com/chem/bioreagents.

Or call your local authorized Agilent distributor.

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